(1) Field of the Invention
The present invention relates to a method of, an apparatus for and a reagent for measuring a catecholamine and its metabolite in a biological sample.
(2) Description of the Prior Art
A measuring error is known to occur due to the presence of a substance which interferences with the fluorescence inducing reaction in the measure of separating catecholamine after the fluorophore containing catecholamine by liquid chromatography and converting the fluorophore of catecholamines in body fluids such as blood, urine and spinal fluid. As a solution of this problem, a method of adding N-ethylmaleimide represented by the following equation is reported (F. A. J. Van der Hoorn et al., Journal of Chromatography, 487, pp 17-28 (1989)). ##STR1##
FIG. 2 illustrates the fluorescent chromatogram of urine analyzed by chromatography. It shows that the peak of catecholamines such as norepinephrine, epinephrine and dopamine contained in the urine is very low. The inhibition of inducing reaction is observed in other body fluids such as blood and spinal fluid.
Said reaction inhibition can be prevented by addition of the substance which would react with the interfering substance in advance and prevent interruption of the fluorescence inducing reaction. However, the conventional additive, N-ethylmaleimide itself causes a fluorescent peak. Therefore, it is very difficult to distinguish between the N-ethylmaleimide fluorescent peak and that of the catecholamine in the biological sample having the retention time adjacent to said peak. This has been a factor causing identification errors, leading to a failure in obtaining correct concentration of the catecholamines. A big peak occurs immediately in the retention time of norepinephrine, one of the catecholamines, especially in the case of the conventional N-ethylmaleimide. It has been found out that the measurements of catecholamines and their metabolites are adversely affected by the peak of 3,4-dihydroxyphenylethyleneglycol (DOPEG), one of the catecholamine metabolites, located contiguous to this position.
Especially, the concentration of catecholamines in the blood plasma is very low, causing a severe impact of the peak given by said interfering substance, and disabling measurement in some cases. Furthermore, the same peak occurs even if the retention time is reduced to some extent, resulting in difficulties, according to a recent finding.